The present invention relates to novel methods for the identification of antigens recognized by cytotoxic T cells (CTLs) and specific for human tumors, cancers, and infected cells, and the use of such antigens in immunogenic compositions or vaccines to induce regression of tumors, cancers, or infections in mammals, including humans. The invention encompasses methods for induction and isolation of cytotoxic T cells specific for human tumors, cancers or infected cells, and for improved selection of genes that encode the target antigens recognized by these specific T cells. The invention also relates to differential display methods that improve resolution of, and that reduce the frequency of false positives of DNA fragments that are differentially expressed in tumorous, cancerous, or infected tissues versus normal tissues. The invention further relates to the engineering of recombinant viruses as expression vectors for tumor, cancer, or infected cell-specific antigens.
Current therapies for cancer include surgery, chemotherapy and radiation. The development and use of immunotherapeutic approaches, e.g., tumor targeting using antibody conjugates, xe2x80x9ccancer vaccinesxe2x80x9d, etc. is an attractive alternative, but has, to date, met with limited success for a number of reasons. The development of monoclonal antibodies specific for tumor antigens, for example, has proved difficult, in part, because antigens that are recognized by monoclonal antibodies and that are expressed by tumors and cancer cells are often also expressed by normal, non-cancerous cells. In addition, the expression of membrane antigens targeted by antibodies is frequently modulated to permit growth of tumor variants that do not express those antigens at the cell surface. A cell-mediated immune response may be more effective for eradication of tumors both because of the different array of effector functions that participate in such responses, and because T cell-mediated responses target not only membrane antigens but any tumor-specific intracellular protein that can be processed and presented in association with major histocompatibility molecules. It is, for this reason, much more difficult for a tumor to evade T cell-surveillance by modulating membrane expression.
Immunotherapeutic approaches based on cell-mediated immune responses are likely to be more effective, but antigens that are expressed by tumors and recognized in cell-mediated immune responses are difficult to identify and to produce. Development of an effective treatment for cancer through vaccination and subsequent stimulation of cell-mediated immunity, has remained elusive; the identification of effective antigens to stimulate cell-mediated responses has been successful only in special cases, such as melanoma. In melanoma, the cytotoxic T cells (CTLs) that mediate a cellular immune response against melanoma infiltrate the tumor itself, and such CTLs can be harvested from the tumor and used to screen for reactivity against other melanoma tumors. Isolation of tumor infiltrating lymphocytes has, however, not been a successful strategy to recover cytotoxic T cells specific for most other tumors, in particular the epithelial cell carcinomas that give rise to greater than 80% of human cancer.
To address the problem of identifying effective antigens for use in vaccination, most previous work has focused on screening expression libraries with tumor-specific CTLs to identify potential tumor antigens. There are significant limitations to the existing methods of identifying effective antigens, including the excessively laborious and inefficient screening process and the considerable difficulty in isolating tumor-specific CTLs for most types of tumors.
Cancer Vaccines
The possibility that altered features of a tumor cell are recognized by the immune system as non-self and may induce protective immunity is the basis for attempts to develop cancer vaccines. Whether or not this is a viable strategy depends on how the features of a transformed cell are altered. Appreciation of the central role of mutation in tumor transformation gave rise to the hypothesis that tumor antigens arise as a result of random mutation in genetically unstable cells. Although random mutations might prove immunogenic, it would be predicted that these would induce specific immunity unique for each tumor. This would be unfavorable for development of broadly effective tumor vaccines. An alternate hypothesis, however, is that a tumor antigen may arise as a result of systematic and reproducible tissue specific gene deregulation that is associated with the transformation process. This could give rise to qualitatively or quantitatively different expression of shared antigens in certain types of tumors that might be suitable targets for immunotherapy. Early results, demonstrating that the immunogenicity of some experimental tumors could be traced to random mutations (De Plaen, et al., Proc. Natl. Acad. Sci. USA 85:2274-2278 (1988); Srivastava, and Old, Immunol. Today 9:78 (1989)), clearly supported the first hypothesis. There is, however, no a priori reason why random mutation and systematic gene deregulation could not both give rise to new immunogenic expression in tumors. Indeed, more recent studies in both experimental tumors (Sahasrabudhe, et al., J. Immunology 151:6202-6310 (1993); Torigoe, et al., J. Immunol. 147:3251 (1991)) and human melanoma (van Der Bruggen, et al., Science 254:1643-1647 (1991); Brichard, et al., J. Exp. Med. 178:489-495 (1993); Kawakami, et al., Proc. Natl. Acad. Sci. USA 91:3515-3519 (1994); Boel, et al., Immunity 2:167-175 (1995); Van den Eynde, et al., J. Exp. Med. 182:689-698 (1995)) have clearly demonstrated expression of shared tumor antigens encoded by deregulated normal genes. The identification of MAGE-1 and other antigens common to different human melanoma holds great promise for the future development of multiple tumor vaccines.
In spite of the progress in melanoma, shared antigens recognized by cytotoxic T cells have not been described for other human tumors. The major challenge is technological. The most widespread and to date most successful approach to identify immunogenic molecules uniquely expressed in tumor cells is to screen a cDNA library with tumor-specific CTLs (cytotoxic T lymphocytes). Application of this strategy has led to identification of several gene families expressed predominantly in human melanoma. Two major limitations of this approach, however, are that (1) screening requires labor intensive transfection of numerous small pools of recombinant DNA into separate target populations in order to assay T cell stimulation by a minor component of some pool; and (2) with the possible exception of renal cell carcinoma, tumor-specific CTLs have been very difficult to isolate from either tumor infiltrating lymphocytes (TIL) or PBL of patients with other types of tumors, especially the epithelial cell carcinomas that comprise greater than 80% of human tumors. It appears that there may be tissue specific properties that result in tumor-specific CTLs being sequestered in melanoma.
Direct immunization with tumor-specific gene products may be essential to elicit an immune response against some shared tumor antigens. It has been argued that, if a tumor expressed strong antigens, it should have been eradicated prior to clinical manifestation. Perhaps then, tumors express only weak antigens. Immunologists have long been interested in the issue of what makes an antigen weak or strong. There have been two major hypotheses. Weak antigens may be poorly processed and fail to be presented effectively to T cells. Alternatively, the number of T cells in the organism with appropriate specificity might be inadequate for a vigorous response (a so-called xe2x80x9chole in the repertoirexe2x80x9d). Elucidation of the complex cellular process whereby antigenic peptides associate with MHC molecules for transport to the cell surface and presentation to T cells has been one of the triumphs of modern immunology. These experiments have clearly established that failure of presentation due to processing defects or competition from other peptides could render a particular peptide less immunogenic. In contrast, it has, for technical reasons, been more difficult to establish that the frequency of clonal representation in the T cell repertoire is an important mechanism of low responsiveness. Recent studies demonstrating that the relationship between immunodominant and cryptic peptides of a protein antigen change in T cell receptor transgenic mice suggest, however, that the relative frequency of peptide-specific T cells can, indeed, be a determining factor in whether a particular peptide is cryptic or dominant in a T cell response. This has encouraging implications for development of vaccines. With present day methods, it would be a complex and difficult undertaking to modify the way in which antigenic peptides of a tumor are processed and presented to T cells. The relative frequency of a specific T cell population can, however, be directly and effectively increased by prior vaccination. This could, therefore, be the key manipulation required to render an otherwise cryptic response immunoprotective.
Another major concern for the development of broadly effective human vaccines is the extreme polymorphism of HLA class I molecules. Class I MHC:cellular peptide complexes are the target antigens for specific CD8+ CTLs. The cellular peptides, derived by degradation of endogenously synthesized proteins, are translocated into a pre-Golgi compartment where they bind to class I MHC molecules for transport to the cell surface. The CD8 molecule contributes to the avidity of the interaction between T cell and target by binding to the xcex13 domain of the class I heavy chain. Since all endogenous proteins turn over, peptides derived from any cytoplasmic or nuclear protein may bind to an MHC molecule and be transported for presentation at the cell surface. This allows T cells to survey a much larger representation of cellular proteins than antibodies which are restricted to recognize conformational determinants of only those proteins that are either secreted or integrated at the cell membrane.
The T cell receptor antigen binding site interacts with determinants of both the peptide and the surrounding MHC. T cell specificity must, therefore, be defined in terms of an MHC:peptide complex. The specificity of peptide binding to MHC molecules is very broad and of relatively low affinity in comparison to the antigen binding sites of specific antibodies. Class I-bound peptides are generally 8-10 residues in length and accommodate amino acid side chains of restricted diversity at certain key positions that match pockets in the MHC peptide binding site. These key features of peptides that bind to a particular MHC molecule constitute a peptide binding motif.
Hence, there exists a need for methods to facilitate the induction and isolation of T cells specific for human tumors, cancers and infected cells and for methods to efficiently select the genes that encode the major target antigens recognized by these T cells in the proper MHC-context.
Vaccinia Vectors
Poxvirus vectors are used extensively as expression vehicles for protein and antigen, e.g. vaccine antigen, expression in eukaryotic cells. Their ease of cloning and propagation in a variety of host cells has led, in particular, to the widespread use of poxvirus vectors for expression of foreign protein and as delivery vehicles for vaccine antigens (Moss, B., Science 252:1662-1667 (1991)).
Customarily, the foreign DNA is introduced into the poxvirus genome by homologous recombination. The target protein coding sequence is cloned behind a vaccinia promoter flanked by sequences homologous to a non-essential region in the poxvirus and the plasmid intermediate is recombined into the viral genome by homologous recombination. This methodology works efficiently for relatively small inserts tolerated by prokaryotic hosts. The method becomes less viable in cases requiring large inserts as the frequency of homologous recombination is low and decreases with increasing insert size; in cases requiring construction of labor intensive plasmid intermediates such as in expression library production; and, in cases where the propagation of DNA is not tolerated in bacteria. Hence, there is a need for improved methods of introducing large inserts at high frequency, that do not require such labor intensive genetic engineering.
Alternative methods using direct ligation vectors have been developed to efficiently construct chimeric genomes in situations not readily amenable for homologous recombination (Merchlinsky, M. et al., Virology 190:522-526 (1992); Scheiflinger, F. et al., Proc. Natl. Acad. Sci. USA. 89:9977-9981 (1992)). These direct ligation protocols have obviated the need for homologous recombination to generate poxvirus chimeric genomes. In such protocols, the DNA from the genome was digested, ligated to insert DNA in vitro, and transfected into cells infected with a helper virus (Merchlinsky, M. et al., Virology 190:522-526 (1992); Scheiflinger, F. et al., Proc. Natl. Acad. Sci. USA 89:9977-9981 (1992)). In one protocol, the genome was digested at the unique NotI site and a DNA insert containing elements for selection or detection of the chimeric genomes was ligated to the genomic arms (Scheiflinger, F. et al., Proc. Natl. Acad. Sci. USA. 89:9977-9981 (1992)). This direct ligation method was described for the insertion of foreign DNA into the vaccinia virus genome (Pfleiderer et al., J. General Virology 76:2957-2962 (1995)). Alternatively, the vaccinia WR genome was modified by removing the NotI site in the HindIII F fragment and reintroducing a NotI site proximal to the thymidine kinase gene such that insertion of a sequence at this locus disrupts the thymidine kinase gene, allowing isolation of chimeric genomes via use of drug selection (Merchlinsky, M. et al., Virology 190:522-526 (1992)).
The direct ligation vector, vNotI/tk allowed one to efficiently clone and propagate DNA inserts at least 26 kilobase pairs in length (Merchlinsky, M. et al., Virology 190:522-526 (1992)). Although, large DNA fragments were efficiently cloned into the genome, proteins encoded by the DNA insert will only be expressed at the low level corresponding to the thymidine kinase gene, a relatively weakly expressed early class gene in vaccinia. In addition, the DNA will be inserted in both orientations at the NotI site. Hence, there is a need for more efficient methods of cloning large DNA fragments into the viral genome with accompanying high levels of expression of the protein product encoded by the DNA insert. There also exists a need for improved direct ligation vectors. Such vectors will be more universally useful for the development of cancer vaccines.
The invention relates to methods for the identification of target antigens recognized by CTLs, and the formulation and use of such antigens in immunogenic compositions or vaccines to induce cell-mediated immunity against target cells, such as tumor cells, that express the target antigens.
Two basic approaches are described for the identification of target antigens. In one approach, CTLs generated against authentic target cells, such as tumor cells, in animals tolerized to non-target (e.g., non-tumorigenic) cellular counterparts are used to screen expression libraries made from target cell-derived (e.g., tumor-derived) DNA, RNA or cDNA to identify clones expressing target antigens. The CTLs generated by the methods described herein are not cross-reactive with normal cells, and thus are better tools for screening. Improved expression libraries are also described.
In a second approach for identifying target antigens, products of genes differentially expressed in target cells, such as tumor cells, are used to immunize animals to generate HLA-restricted CTLs which are evaluated for activity against authentic target cells. Like the first approach, this second strategy could also be particularly useful for identifying epitopes for many human tumor types where it has not been possible to generate tumor-specific CTLs directly from patients. In addition, it may identify cryptic antigens of the intact tumor cellxe2x80x94i.e., tumor cell products which can become immunogenic, if the representation of tumor-specific CTLs is first augmented by vaccination with that tumor cell product. Modified methods for differential display that improve resolution and reduce false positives are described.
In accordance with the present invention, the target cell is a cell to which it is desirable to induce a cell-mediated immune response. Examples of target cells in the body include, but are not limited to, tumor cells, malignant cells, transformed cells, cells infected with a virus, fungus, or mycobacteria, or cells subject to any other disease condition which leads to the production of target antigens.
The invention also encompasses the high yield expression of candidate target antigens, and production of recombinant viruses for vaccine formulation.
Abbreviations
CTLsxe2x80x94cytotoxic T lymphocytes (T cells)
PBLxe2x80x94peripheral blood lymphocytes
RDAxe2x80x94Representational Difference Analysis
TILxe2x80x94tumor infiltrating lymphocytes